Cell culture and SLC12A1 intervene

HEK293T cells were maintained in RPMI-1640 with 10% FBS. HCC cells were maintained in Dulbecco's modified Eaglemedium (DMEM) with 10% FBS, 1 mM glutamine. As for siRNA transfection, cells were seeded into plates and incubated overnight, and then transfected with RNAs using Oligofectamine™ (Invitrogen CA, USA). The cells were culturedand collected at 0, 24 and 48 h after transfection. Non-targeting siRNA with the following sequence was applied as a control: sense: UUCUCCGAACGUGUCACGUTT, anti-sense: ACGUGACACGUUCGGAGAATT. Three siRNA of SLC12A1 with the following sequences were designed: si-1: sense-CGCGCAAACUGUGUGUUAATT, antisense-UUAACACACAGUUUGCGCGTT; si-2: sense -GCUGGCAAGUUGAACAUUATT, antisense-UAAUG UUCAACUUGCAGCTT; si-3: sense-GUCCAGAGGUUU CUUUAAUTT, antisense-AUUAAAGAAACCUCUGGA CTT.

Lentivirus packed with dCas9-VP64 and MS2-P65-HSF1(SAM) was purchased from Hanbio Biotechnology (Shanghai, China). Lentivirus packed with dCas9-KRAB was purchased from YSY Biotechnology (Nanjing, China). The sgRNA was designed online at GENSCRIPT (http://www.genscript.com/gRNA-database), and had the following the sequence: sgSLC12A1-1: CAACTT AAGTACCACATCAAC; sgSLC12A1-2: CCTCAGGGC TAATGACCTGG; sgSLC12A1-3: GGGGCTTACAG TACTATTCC. The sgRNA was synthesized at Genebioseq (Shanghai, China) and assembled into restriction BsmBI sites of lentiGuide-Puro(Addgene plasmid 52963, a gift from Feng Zhang) following Zhang et al.'s protocol [47]. Recombinant plasmids (10μg) were cotransfected into HEK293T with lentiviral packaging vectors pRSV-REV (5 μg), pMDLg/pRRE (10 μg) and vesicular stomatitis virus G glycoprotein (VSVG) expression vector pMD2G (7.5 μg). After three days, the post-transfection culture supernatant was concentrated by ultracentrifugation for 1 h at 30,000 rpm, then resuspended in Hank's buffer. The titer was 5~10⁷IU/mL.

The candidate cells were infected at 10 MOI of dCas9-VP64 or dCas9-KRAB and 5 MOI of each sgRNA in the presence of polybrene (6μg/mL), then cultured for further experiments.